Hu L, Olson A J, Weiner R I, Goldsmith P C
Department of Obstetrics and Gynecology, Reproductive Endocrinology Center, San Francisco, Calif., USA.
Neuroendocrinology. 1999 Oct;70(4):221-7. doi: 10.1159/000054480.
Gap junctions (GJs) are transmembrane channels that permit rapid intercellular transit of various small molecules including ions, second messengers and metabolites. GJs promote communication and coordinated activity between coupled neurons, and may help facilitate the synchronous release and pulsatile secretion of neurohormones. A previous study using GnRH-secreting GT1-7 cells reported that connexin 26 was the major GJ subunit present, and that about 20% of the cultured cells engaged in GJ coupling as assayed by fluorescence recovery after photobleaching of 5,6-carboxyfluorescein diacetate (MW 460 D). To reassess GJ connectivity with a more permeant probe, we grew GT1-7 cells to 70% confluency on Matrigel-coated glass coverslips and microinjected Neurobiotin(TM) (MW 322 D) into single cells. Dye was allowed to diffuse for 30 min before cultures were fixed, and subsequently immunostained for Neurobiotin with 3,3'-diaminobenzidine HCl and examined by light microscopy. Dye coupling between 2 or more GT1-7 cells was observed after 75% of all microinjections. Connectivity involved the somata and neurites of an average of 6.6 +/- 2.0 adjoining cells, but in one instance was seen in a group of 32 GT1-7 neighbors. Western blotting and immunofluorescence staining confirmed that connexin 26 was the predominant GJ subunit expressed by GT1-7 cultures. Our results using Neurobiotin suggest these GJ channels may be smaller than anticipated. In addition, functional GJ connectivity between subconfluent GT1-7 cells is more extensive than previously reported, occurring with higher frequency and coupling significantly greater numbers of cultured cells. Since cAMP, IP3, and Ca(2+) are able to pass through GJs and can elicit secretion of GnRH by GT1 cell cultures, GJs may play an important role in the coordination and synchronization of GnRH release.
缝隙连接（GJs）是跨膜通道，可允许包括离子、第二信使和代谢物在内的各种小分子在细胞间快速转运。缝隙连接促进耦合神经元之间的通讯和协调活动，并可能有助于促进神经激素的同步释放和脉冲式分泌。先前一项使用分泌促性腺激素释放激素（GnRH）的GT1-7细胞的研究报告称，连接蛋白26是主要存在的缝隙连接亚基，并且通过5,6-羧基荧光素二乙酸酯（分子量460 D）光漂白后的荧光恢复测定，约20%的培养细胞参与了缝隙连接耦合。为了用更具渗透性的探针重新评估缝隙连接的连通性，我们将GT1-7细胞在基质胶包被的玻璃盖玻片上培养至70%汇合度，并将神经生物素（TM）（分子量322 D）显微注射到单个细胞中。在培养物固定前让染料扩散30分钟，随后用3,3'-二氨基联苯胺盐酸盐对神经生物素进行免疫染色，并通过光学显微镜检查。在所有显微注射的75%之后观察到两个或更多GT1-7细胞之间的染料耦合。连通性涉及平均6.6±2.0个相邻细胞的胞体和神经突，但在一个实例中在一组32个GT1-7相邻细胞中观察到。蛋白质免疫印迹和免疫荧光染色证实连接蛋白26是GT1-7培养物中表达的主要缝隙连接亚基。我们使用神经生物素的结果表明这些缝隙连接通道可能比预期的小。此外，亚汇合GT1-7细胞之间的功能性缝隙连接连通性比先前报道的更广泛，发生频率更高，耦合的培养细胞数量显著更多。由于环磷酸腺苷（cAMP）、肌醇三磷酸（IP3）和钙离子（Ca(2+)）能够通过缝隙连接并能引发GT1细胞培养物中GnRH的分泌，缝隙连接可能在GnRH释放的协调和同步中起重要作用。
